Combination of an anti-cd19 antibody and a bruton&#39;s tyrosine kinase inhibitor and uses thereof

ABSTRACT

The present disclosure describes a pharmaceutical combination of an anti-CD19 antibody and a Bruton&#39;s tyrosine kinase (BTK) inhibitor and its use for the treatment of non-Hodgkin&#39;s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia.

This patent application is a continuation of U.S. patent applicationSer. No. 15/572,914 filed Nov. 9, 2017, which is the U.S. National Stageof International Application No. PCT/EP2016/061744 filed May 25, 2016,which claims the benefit of EP 15196860.9 filed Nov. 27, 2015 and EP15169124.3 filed May 26, 2015, the content of each of which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure is related to a pharmaceutical combination of ananti-CD19 antibody and a Bruton's tyrosine kinase (BTK) inhibitor forthe treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemiaand/or acute lymphoblastic leukemia.

BACKGROUND

B cells are lymphocytes that play a large role in the humoral immuneresponse. They are produced in the bone marrow of most mammals, andrepresent 5-15% of the circulating lymphoid pool. The principal functionof B cells is to make antibodies against various antigens, and are anessential component of the adaptive immune system.

Because of their critical role in regulating the immune system,disregulation of B cells is associated with a variety of disorders, suchas lymphomas, and leukemias. These include non-Hodgkin's lymphoma (NHL),chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia(ALL).

NHL is a heterogeneous malignancy originating from lymphocytes. In theUnited States (U.S.), the incidence is estimated at 65,000/year withmortality of approximately 20,000 (American Cancer Society, 2006; andSEER Cancer Statistics Review). The disease can occur in all ages, theusual onset begins in adults over 40 years, with the incidenceincreasing with age. NHL is characterized by a clonal proliferation oflymphocytes that accumulate in the lymph nodes, blood, bone marrow andspleen, although any major organ may be involved. The currentclassification system used by pathologists and clinicians is the WorldHealth Organization (WHO) Classification of Tumours, which organizes NHLinto precursor and mature B-cell or T-cell neoplasms. The PDQ iscurrently dividing NHL as indolent or aggressive for entry into clinicaltrials. The indolent NHL group is comprised primarily of follicularsubtypes, small lymphocytic lymphoma, MALT (mucosa-associated lymphoidtissue), and marginal zone; indolent encompasses approximately 50% ofnewly diagnosed B-cell NHL patients. Aggressive NHL includes patientswith histologic diagnoses of primarily diffuse large B cell (DLBL,DLBCL, or DLCL) (40% of all newly diagnosed patients have diffuse largecell), Burkitt's, and mantle cell. The clinical course of NHL is highlyvariable. A major determinant of clinical course is the histologicsubtype. Most indolent types of NHL are considered to be incurabledisease. Patients respond initially to either chemotherapy or antibodytherapy and most will relapse. Studies to date have not demonstrated animprovement in survival with early intervention. In asymptomaticpatients, it is acceptable to “watch and wait” until the patient becomessymptomatic or the disease pace appears to be accelerating. Over time,the disease may transform to a more aggressive histology. The mediansurvival is 8 to 10 years, and indolent patients often receive 3 or moretreatments during the treatment phase of their disease. Initialtreatment of the symptomatic indolent NHL patient historically has beencombination chemotherapy. The most commonly used agents include:cyclophosphamide, vincristine and prednisone (CVP); or cyclophosphamide,adriamycin, vincristine, prednisone (CHOP). Approximately 70% to 80% ofpatients will respond to their initial chemotherapy, duration ofremissions last on the order of 2-3 years. Ultimately the majority ofpatients relapse. The discovery and clinical use of the anti-CD20antibody, rituximab, has provided significant improvements in responseand survival rate. The current standard of care for most patients isrituximab+CHOP (R—CHOP) or rituximab+CVP (R—CVP). Interferon is approvedfor initial treatment of NHL in combination with alkylating agents, buthas limited use in the U.S. Rituximab therapy has been shown to beefficacious in several types of NHL, and is currently approved as afirst line treatment for both indolent (follicular lymphoma) andaggressive NHL (diffuse large B cell lymphoma). However, there aresignificant limitations of anti-CD20 monoclonal antibody (mAb),including primary resistance (50% response in relapsed indolentpatients), acquired resistance (50% response rate upon re-treatment),rare complete response (2% complete resonse rate in relapsedpopulation), and a continued pattern of relapse. Finally, many B cellsdo not express CD20, and thus many B-cell disorders are not treatableusing anti-CD20 antibody therapy.

In addition to NHL there are several types of leukemias that result fromdisregulation of B cells. Chronic lymphocytic leukemia (also known as“chronic lymphoid leukemia” or “CLL”), is a type of adult leukemiacaused by an abnormal accumulation of B lymphocytes. In CLL, themalignant lymphocytes may look normal and mature, but they are not ableto cope effectively with infection. CLL is the most common form ofleukemia in adults. Men are twice as likely to develop CLL as women.However, the key risk factor is age. Over 75% of new cases are diagnosedin patients over age 50. More than 10,000 cases are diagnosed every yearand the mortality is almost 5,000 a year (American Cancer Society, 2006;and SEER Cancer Statistics Review). CLL is an incurable disease butprogresses slowly in most cases. Many people with CLL lead normal andactive lives for many years. Because of its slow onset, early-stage CLLis generally not treated since it is believed that early CLLintervention does not improve survival time or quality of life. Instead,the condition is monitored over time. Initial CLL treatments varydepending on the exact diagnosis and the progression of the disease.There are dozens of agents used for CLL therapy. Combinationchemotherapy regimens such as FCR (fludarabine, cyclophosphamide andrituximab), and BR (Ibrutinib and rituximab) are effective in bothnewly-diagnosed and relapsed CLL. Allogeneic bone marrow (stem cell)transplantation is rarely used as a first-line treatment for CLL due toits risk.

Another type of leukemia is acute lymphoblastic leukemia (ALL), alsoknown as acute lymphocytic leukemia. ALL is characterised by theoverproduction and continuous multiplication of malignant and immaturewhite blood cells (also known as lymphoblasts) in the bone marrow.‘Acute’ refers to the undifferentiated, immature state of thecirculating lymphocytes (“blasts”), and that the disease progressesrapidly with life expectancy of weeks to months if left untreated. ALLis most common in childhood with a peak incidence of 4-5 years of age.Children of age 12-16 die more easily from it than others. Currently, atleast 80% of childhood ALL are considered curable. Under 4,000 cases arediagnosed every year and the mortality is almost 1,500 a year (AmericanCancer Society, 2006; and SEER Cancer Statistics Review).

The human CD 19 molecule is a structurally distinct cell surfacereceptor expressed on the surface of human B cells, including, but notlimited to, pre-B cells, B cells in early development {i.e., immature Bcells), mature B cells through terminal differentiation into plasmacells, and malignant B cells. CD 19 is expressed by most pre-B acutelymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B cell chroniclymphocytic leukemias (CLL), pro-lymphocytic leukemias, hairy cellleukemias, common acute lymphocytic leukemias, and some Null-acutelymphoblastic leukemias (Nadler et al, J. Immunol., 131:244-250 (1983),Loken et al, Blood, 70:1316-1324 (1987), Uckun et al, Blood, 71:13-29(1988), Anderson et al, 1984. Blood, 63:1424-1433 (1984), Scheuermann,Leuk. Lymphoma, 18:385-397(1995)). The expression of CD 19 on plasmacells further suggests it may be expressed on differentiated B celltumors such as multiple myeloma, plasmacytomas, Waldenstrom's tumors(Grossbard et al., Br. J. Haematol, 102:509-15(1998); Treon et al,Semin. Oncol, 30:248-52(2003)).

Therefore, the CD 19 antigen is a target for immunotherapy in thetreatment of non-Hodgkin's lymphoma (including each the subtypesdescribed herein), chronic lymphocytic leukemia and/or acutelymphoblastic leukemia.

Certain CD19 therapies have been shown. T cells expressing an anti-CD19chimeric antigen receptor (CAR) including both CD3-ζ and the 4-BBcostimulatory domain were administered to three patients with advancedCLL. Kalos et al., T cells with Chimeric Antigen Receptors Have PotentAntitumor Effects and Can Establish Memory in Patients with AdvancedLeukemia, Science Translational Medicine, vol. 3, no. 95 (10 Aug. 2011),which is incorporated by reference in its entirety. Sadelain et al., Thepromise and potential pitfalls of chimeric antigen receptors, CurrentOpinion in Immunology, Elsevier, vol. 21, no. 2, 2 Apr. 2009, which isincorporated by reference in its entirety, also describes anti-CD19chimeric antigen receptors (CARs). Neither Kalos et al. nor Sadelain etal., however, describe the antibody specific for CD19 in combinationwith a Bruton's tyrosine kinase (BTK) inhibitor as exemplified herein.

The use of a CD19 antibody in non-specific B cell lymphomas is discussedin WO2007076950 (US2007154473), which are both incorporated by referencein their entireties, along with the cursory mention of Ibrutinib withina long list of potential combination partners, but fails either to teachthe antibody exemplified herein or suggest the synergistic effects ofthe combination in the treatment of non-Hodgkin's lymphoma, chroniclymphocytic leukemia and/or acute lymphoblastic leukemia as exemplifiedherein.

The use of a CD19 antibody in CLL, NHL and ALL is described inScheuermann et al., CD19 Antigen in Leukemia and Lymphoma Diagnosis andImmunotherapy, Leukemia and Lymphoma, Vol. 18, 385-397 (1995), which isincorporated by reference in its entirety, but fails to suggest thecombination exemplified herein.

Additional antibodies specific for CD19 are described in WO2005012493(U.S. Pat. No. 7,109,304), WO2010053716 (U.S. Ser. No. 12/266,999)(Immunomedics); WO2007002223 (U.S. Pat. No. 8,097,703) (Medarex);WO2008022152 (Ser. No. 12/377,251) and WO2008150494 (Xencor),WO2008031056 (U.S. Ser. No. 11/852,106) (Medimmune); WO 2007076950 (U.S.Ser. No. 11/648,505) (Merck Patent GmbH); WO 2009/052431 (U.S. Ser. No.12/253,895) (Seattle Genetics); and WO2010095031 (Ser. No. 12/710,442)(Glenmark Pharmaceuticals), WO2012010562 and WO2012010561 (InternationalDrug Development), WO2011147834 (Roche Glycart), and WO 2012/156455(Sanofi), which are all incorporated by reference in their entireties.

Combinations of antibodies specific for CD19 and other agents aredescribed in WO2010151341 (U.S. Ser. No. 13/377,514) (The FeinsteinInstitute); U.S. Pat. No. 5,686,072 (University of Texas), andWO2002022212 (PCT/US01/29026) (IDEC Pharmaceuticals), WO2013/024097(Ser. No. 14/126,928) (MorphoSys AG) and WO2013/024095 (Ser. No.14/127,217) (MorphoSys AG), which are all incorporated by reference intheir entireties.

Certain bruton's tyrosine kinase inhibitors are commercially available.Ibrutinib, also known as PCI-32765, and marketed under the nameImbruvica is an anticancer drug targeting B-cell malignancies. Ibrutinibis described in U.S. Pat. Nos. 7,514,444; 8,008,309; 8,697,711;8,735,403; 8,957,079; and 8,754,090, which are all incorporated byreference in their entirities.

Ibrutinib has been tested in combination with rituximab (an anti-CD20antibody). Burger et al., Lancet Oncol., 2014 September, 15(19):1090-1099. Ibrutinib potentially inhibits the ADCC activity of certainCD20 antibodies in vitro. Duong et al., mAbs, January/February 2015,192-198, and Kohrt et al., Blood, 2014, 123:1957-1960.

It is clear that in spite of the recent progress in the discovery anddevelopment of anticancer agents, many forms of cancer involvingCD19-expressing tumors still have a poor prognosis. Thus, there is aneed for improved methods for treating such forms of cancer.

SUMMARY

Neither alone nor in combination does the prior art suggest thesynergistic effects of the combination of the exemplified antibody andIbrutinib in the treatment of non-Hodgkin's lymphoma, chroniclymphocytic leukemia and/or acute lymphoblastic leukemia.

In one aspect, the present disclosure relates to a synergisticcombination of an antibody specific for CD19 and a Bruton's tyrosinekinase (BTK) inhibitor. Such combinations are useful in the treatment ofB cell malignancies, such as, non-Hodgkin's lymphoma, chroniclymphocytic leukemia and/or acute lymphoblastic leukemia.

In vitro models are considered indicative of how a certain compound orcombination of compounds would behave in humans.

MEC-1 cells in this in vitro model are indicative of how the combinationwill work in the treatment of chronic lymphoid leukemia (CLL) in humans.Ramos cells in this in vitro model are indicative of how the combinationwill work in the treatment of non-Hogkins lymphoma (NHL) in humans.MEC-1 cells (DSMZ # ACC497) are a chronic B-cell leukemia cell line.Ramos cells (ATCC number CRL-1596), a human Burkitt's lymphoma cells.

In addition, when compounds are combined in vitro, one expects that thecombination has only additive effects. Surprisingly, the inventors foundthat the combination of a particular antibody specific for CD19 andIbrutinib mediated a synergistic level of specific cell killing in vitroin comparison to the antibody and Ibrutinib alone. Specifically, theinventors found that the combination of MOR00208 and Ibrutinib mediateda synergistic level of specific cell killing in vitro in MEC-1 cellscompared to the antibody and Ibrutinib alone.

In addition, and also unexpectedly, the inventors found that thecombination of a particular antibody specific for CD19 and Ibrutinib hadcertain functional properties, in comparison to the antibody andIbrutinib alone.

In summary, the combination of the exemplified anti-CD19 antibody andIbrutinib behaved synergistically in models relevant to CLL. As CLL is aB cell related disorders and CD19 is highly expressed on B-cells, theexemplified combination would have the same mechanism of action andshould also behave synergistically in the treatment of other B cellrelated disorders, e.g. ALL and NHL.

Therefore, the combination of the exemplified antibody specific for CD19and Ibrutinib should be effective in the treatment of humans innon-Hodgkin's lymphoma, chronic lymphocytic leukemia and/or acutelymphoblastic leukemia. The expected efficacy of the combination of theantibody specific to CD19 exemplified and Ibrutinib will be confirmed inclinical trials.

As the mechanism of action of Ibrutinib and other Bruton's tyrosinekinase (BTK) inhibitors are similar, as they all work by inhibiting thetyrosine-protein kinase BTK enzyme, which plays a crucial role in B-celldevelopment, it is believed that synergy should also be seen whentreating humans having non-Hodgkin's lymphoma, chronic lymphocyticleukemia and/or acute lymphoblastic leukemia with a combination of theexemplified anti-CD19 antibody and a Bruton's tyrosine kinase (BTK)inhibitor other than Ibrutinib.

As the exemplified anti-CD19 antibody and other anti-CD19 antibodiesbind CD19, it is believed that synergy should also be seen when treatinghumans having non-Hodgkin's lymphoma, chronic lymphocytic leukemiaand/or acute lymphoblastic leukemia with a combination of any anti-CD19antibody and a Bruton's tyrosine kinase (BTK) inhibitor, e.g.,Ibrutinib.

An aspect of the present disclosure comprises a synergistic combinationwherein the antibody specific for CD19 comprises an HCDR1 region ofsequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), anLCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region ofsequence MQHLEYPIT (SEQ ID NO: 6) and Ibrutinib. In preferred aspects,the combination is used for the treatment of non-Hodgkin's lymphoma,chronic lymphocytic leukemia and/or acute lymphoblastic leukemia.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the cytotoxicity effects of MOR00208 and Ibrutinib aloneand in combination on Ramos cells. Ramos cells were pre-treated with 40μM Ibrutinib for 24 hours.

FIG. 2 shows the cytotoxicity effects of MOR00208 and Ibrutinib aloneand in combination on MEC-1 cells. MEC-1 cells were pre-treated with 30μM Ibrutinib for 24 hours.

FIG. 3 shows the cytotoxicity effects of MOR00208 and Ibrutinib aloneand in combination on MEC-1 cells. MEC-1 cells were pre-treated with 30μM Ibrutinib for 24 hours.

FIG. 4 shows the amino acid sequence of the variable domains ofMOR00208.

FIG. 5 shows the amino acid sequence of the Fc regions of MOR00208.

FIG. 6, FIG. 7 and FIG. 8 show the cytotoxicity of MOR00208 andIbrutinib alone and in combination in MEC-1 cell line.

FIG. 9, FIG. 10 and FIG. 11 show Chou-Talay Combination Index curves ofMOR00208 and Ibrutinib in combination in MEC-1 cell line.

FIG. 12, FIG. 13, FIG. 14, FIG. 15, FIG. 16, FIG. 17, FIG. 18, FIG. 19and FIG. 20 show the Clarke et al. synergy calculations of MOR00208 andIbrutinib in combination in MEC-1 cell line.

DETAILED DESCRIPTION OF THE INVENTION

“Synergy”, “synergism” or “synergistic” mean more than the expectedadditive effect of a combination. The “synergy”, “synergism” or“synergistic” effect of a combination is determined herein by themethods of Chou et al., Clarke et al. and/or Webb et al. See Ting-ChaoChou, Theoretical Basis, Experimental Design, and ComputerizedSimulation of Synergism and Antagonism in Drug Combination Studies,Pharmacol Rev 58:621-681 (2006), which is incorporated by reference inits entirety. See also Clarke et al., Issues in experimental design andendpoint analysis in the study of experimental cytotoxic agents in vivoin breast cancer and other models, Breast Cancer Research and Treatment46:255-278 (1997), which is incorporated by reference in its entirety.See also Webb, J. L. (1963) Enzyme and Metabolic Inhibitors, AcademicPress, New York, which is incorporated by reference in its entirety.

The term “antibody” means monoclonal antibodies, including any isotype,such as, IgG, IgM, IgA, IgD and IgE. An IgG antibody is comprised of twoidentical heavy chains and two identical light chains that are joined bydisulfide bonds. Each heavy and light chain contains a constant regionand a variable region. Each variable region contains three segmentscalled “complementarity-determining regions” (“CDRs”) or “hypervariableregions”, which are primarily responsible for binding an epitope of anantigen. They are referred to as CDR1, CDR2, and CDR3, numberedsequentially from the N-terminus. The more highly conserved portions ofthe variable regions outside of the CDRs are called the “frameworkregions”. An “antibody fragment” means an Fv, scFv, dsFv, Fab, Fab′F(ab′)2 fragment, or other fragment, which contains at least onevariable heavy or variable light chain, each containing CDRs andframework regions.

A “Bruton's tyrosine kinase (BTK) inhibitor” is a class of drug thatfunctions by inhibiting the tyrosine-protein kinase BTK enzyme, whichplays an important role in B-cell development. Specifically, BTKcontains a PH domain that binds phosphatidylinositol(3,4,5)-trisphosphate (PIP3). PIP3 binding induces Btk to phosphorylatephospholipase C, which in turn hydrolyzes PIP2, a phosphatidylinositol,into two second messengers, inositol triphosphate (IP3) anddiacylglycerol (DAG), which then go on to modulate the activity ofdownstream proteins during B-cell signalling.

Bruton's tyrosine kinase (BTK) inhibitors include Ibrutinib. Ibrutinibis marketed by Pharmacyclics, Inc and Johnson & Johnson's JanssenPharmaceutical (trade name Imbruvica, also named PCI-32765). Ibrutinibis is currently labelled for the treatment of patients with Mantle celllymphoma (MCL) who have received at least one prior therapy, Chroniclymphocytic leukemia (CLL) who have received at least one prior therapy,Chronic lymphocytic leukemia with 17p deletion, and Waldenstrom'smacroglobulinemia. The formula of Ibrutinib is1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-oneand has the following structure:

Ibrutinib is a small-molecule inhibitor of BTK. Ibrutinib forms acovalent bond with a cysteine residue in the BTK active site, leading toinhibition of BTK enzymatic activity. BTK is a signaling molecule of theB-cell antigen receptor (BCR) and cytokine receptor pathways. BTK's rolein signaling through the B-cell surface receptors results in activationof pathways necessary for B-cell trafficking, chemotaxis, and adhesion.Nonclinical studies show that ibrutinib inhibits malignant B-cellproliferation and survival in vivo as well as cell migration andsubstrate adhesion in vitro.

Additional Bruton's tyrosine kinase (BTK) inhibitors include:

ACP-196 (Acerta Pharma BV), which is described in WO 2012170976 (U.S.application Ser. No. 14/112,428), WO 2013010380 (U.S. application Ser.No. 14/233,478), and WO 2014113932 (U.S. application Ser. No.14/160,587), which are all incorporated by reference in theirentireties;

BGB-3111 (BeiGene, Co., Ltd.), and CC-292 Evans et al. 2013

“VH” refers to the variable region of an immunoglobulin heavy chain ofan antibody, or antibody fragment. “VL” refers to the variable region ofthe immunoglobulin light chain of an antibody, or antibody fragment.

The term “CD19” refers to the protein known as CD19, having thefollowing synonyms: B4, B-lymphocyte antigen CD19, B-lymphocyte surfaceantigen B4, CVID3, Differentiation antigen CD19, MGC12802, and T-cellsurface antigen Leu-12.

Human CD19 has the amino acid sequence of:

(SEQ ID NO: 7) MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRM GTWSTR.

“MOR00208” is an anti-CD19 antibody. The amino acid sequence of thevariable domains is provided in FIG. 4. The amino acid sequence of theheavy and light chain Fc regions of MOR00208 are provided in FIG. 5.“MOR00208” and “XmAb 5574” are used as synonyms to describe the antibodyshown in FIGS. 4 and 5. The MOR00208 antibody is described in U.S.patent application Ser. No. 12/377,251, which is incorporated byreference in its entirety.

U.S. patent application Ser. No. 12/377,251 describes the antibody named4G7 H1.52 Hybrid S239D/1332E/4G7 L1.155 (later named MOR00208) asfollows:

>4G7 H1.52 Hybrid S239D/I332E (SEQ ID NO: 14)EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K >4G7 L1.155(SEQ ID NO: 15) DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

Additional antibodies specific for CD19 are described in U.S. Pat. No.7,109,304 (Immunomedics), which is incorporated by reference in itsentirety; U.S. application Ser. No. 11/917,750 (Medarex), which isincorporated by reference in its entirety; U.S. application Ser. No.11/852,106 (Medimmune), which is incorporated by reference in itsentirety; U.S. application Ser. No. 11/648,505 (Merck Patent GmbH),which is incorporated by reference in its entirety; U.S. Pat. No.7,968,687 (Seattle Genetics), which is incorporated by reference in itsentirety; and U.S. application Ser. No. 12/710,442 (GlenmarkPharmaceuticals), which is incorporated by reference in its entirety.

“Fc region” means the constant region of an antibody, which in humansmay be of the IgG1, 2, 3, 4 subclass or others. The sequences of humanFc regions are available at IMGT, Human IGH C-REGIONs,http://www.imgt.org/IMGTrepertoire/Proteins/protein/human/IGH/IGHC/Hu_IGHCallgenes.html(retrieved on 16 May 2011).

“RefmAb33” is an antibody whose amino acid sequence is as follows:

Heavy chain including the Fc region: (SEQ ID NO: 8)QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLight chain including the Fc region: (SEQ ID NO: 9)DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC

RefmAb33 is specific for RSV, and is used as isotype control, as itshares the same Fc region as MOR00208.

A “combination” means more than one item, e.g. a compound such as anantibody and Ibrutinib.

The present disclosure also relates to combinations, pharmaceuticals,and pharmaceutical compositions containing the described combinations.The two components of the synergistic combination of the presentinvention, e.g. the antibody specific for CD19 and Ibrutinib, may beadministered together, simultaneously, separately or subsequently,either physically or in time.

Ibrutinib is currently taken orally and is currently dosed once per day.MOR00208 is currently administered intravenously, and is currently dosedeither once a week or once every two weeks.

Preferably, administration of both drugs allows for both drugs to beactive in the patent at the same time. For example, if MOR208 is dosedweekly and Ibritinib is dosed daily then the active substance of bothdrugs is present in the patient at the same time. In an embodiment,Ibrutinib, is administered prior to and/or separately from theadministration of the antibody specific for CD19, e.g. MOR00208.

Simultaneously means that the two components are administered at a timewhere both components (drugs) are active in the patient at the sametime. It is implied by “synergism” that both drugs are active in thepatient at the same time.

Administered together can mean administered at the same time.

The two components may be formulated in different pharmaceuticalcompositions. A pharmaceutical composition includes an active agent, eg.an antibody for therapeutic use in humans. A pharmaceutical compositionmay include acceptable carriers or excipients.

“Administered” or “administration” includes but is not limited todelivery by an injectable form, such as, for example, an intravenous,intramuscular, intradermal or subcutaneous route or mucosal route, forexample, as a nasal spray or aerosol for inhalation or as an ingestablesolution, capsule or tablet.

A “therapeutically effective amount” of a compound or combination refersto an amount sufficient to cure, alleviate or partially arrest theclinical manifestations of a given disease or disorder and itscomplications. The amount that is effective for a particular therapeuticpurpose will depend on the severity of the disease or injury as well ason the weight and general state of the subject. It will be understoodthat determination of an appropriate dosage may be achieved, usingroutine experimentation, by constructing a matrix of values and testingdifferent points in the matrix, all of which is within the ordinaryskills of a trained physician or clinical scientist.

The “CDRs” herein are defined by either Chothia et al or Kabat et al.See Chothia C, Lesk AM. (1987) Canonical structures for thehypervariable regions of immunoglobulins. J Mol Biol., 196(4):901-17,which is incorporated by reference in its entirety. See Kabat E. A, WuT. T., Perry H. M., Gottesman K. S. and Foeller C. (1991). Sequences ofProteins of Immunological Interest. 5th edit., NIH Publication no.91-3242, US Dept. of Health and Human Services, Washington, D.C., whichis incorporated by reference in its entirety.

“Cross competes” means the ability of an antibody or other binding agentto interfere with the binding of other antibodies or binding agents toCD19 in a standard competitive binding assay. The ability or extent towhich an antibody or other binding agent is able to interfere with thebinding of another antibody or binding molecule to CD19, and, thereforewhether it can be said to cross-compete according to the invention, canbe determined using standard competition binding assays. One suitableassay involves the use of the Biacore technology (e.g. by using theBIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measurethe extent of interactions using surface plasmon resonance technology.Another assay for measuring cross-competing uses an ELISA-basedapproach. A high throughput process for “epitope binning” antibodiesbased upon their cross-competition is described in International PatentApplication No. WO 2003/48731

The term “epitope” includes any protein determinant capable of specificbinding to an antibody or otherwise interacting with a molecule.Epitopic determinants generally consist of chemically active surfacegroupings of molecules such as amino acids or carbohydrate or sugar sidechains and can have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics. An epitopemay be “linear” or “conformational.” The term “linear epitope” refers toan epitope with all of the points of interaction between the protein andthe interacting molecule (such as an antibody) occur linearally alongthe primary amino acid sequence of the protein (continuous). The term“conformational epitope” refers to an epitope in which discontinuousamino acids that come together in three dimensional conformation. In aconformational epitope, the points of interaction occur across aminoacid residues on the protein that are separated from one another.

“Binds the same epitope as” means the ability of an antibody or otherbinding agent to bind to CD19 and having the same epitope as theexemplified antibody. The epitopes of the exemplified antibody and otherantibodies to CD19 can be determined using standard epitope mappingtechniques. Epitope mapping techniques, well known in the art. includeEpitope Mapping Protocols in Methods in Molecular Biology, Vol. 66(Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example,linear epitopes may be determined by e.g., concurrently synthesizinglarge numbers of peptides on solid supports, the peptides correspondingto portions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al, (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002;Geysen et al, (1985) Proc. Natl. Acad. Sci. USA 82:78-182; Geysen et al,(1986) Mol. Immunol. 23:709-715. Similarly, conformational epitopes arereadily identified by determining spatial conformation of amino acidssuch as by, e.g., hydrogen/deuterium exchange, x-ray crystallography andtwo-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Antigenic regions of proteins can also be identifiedusing standard antigenicity and hydropathy plots, such as thosecalculated using, e.g., the Omiga version 1.0 software program availablefrom the Oxford Molecular Group. This computer program employs theHopp/Woods method, Hopp et al, (1981) Proc. Natl. Acad. Sci USA78:3824-3828; for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al, (1982) J. Mol. Biol. 157: 105-132;for hydropathy plots.

Embodiments

An aspect of the present disclosure is a combination comprising anantibody specific for CD19 and a Bruton's tyrosine kinase (BTK)inhibitor for use in the treatment of non-Hodgkin's lymphoma, chroniclymphocytic leukemia and/or acute lymphoblastic leukemia. Inembodiments, the combination is synergistic.

Herein, the combination of the exemplified anti-CD19 antibody andIbrutinib behaved synergistically in in vitro models relevant to CLL. AsCLL is a B cell related disorder and CD19 is highly expressed onB-cells, the exemplified combination should have the same mechanism ofaction and should also behave synergistically in the treatment of otherB cell related disorders, e.g. ALL and NHL. Therefore, the combinationof the exemplified antibody specific for CD19 and Ibrutinib should beeffective in the treatment of humans in non-Hodgkin's lymphoma, chroniclymphocytic leukemia and/or acute lymphoblastic leukemia. The expectedefficacy of the combination of the antibody specific to CD19 exemplifiedand Ibrutinib will be confirmed in clinical trials.

MEC-1 cells (DSMZ # ACC497) a chronic B-cell leukemia cell line wastested. MEC-1 cells in this in vitro model are indicative of how thecombination will work in the treatment of chronic lymphoid leukemia(CLL) in humans. The Chou index values indicate clear synergism of thecombination of MOR00208 and Ibrutinib in the specific killing of MEC-1cells as compared to MOR00208 and Ibrutinib alone.

Additional cell lines are evaluated: Ramos cells (ATCC number CRL-1596),a human Burkitt's lymphoma cells. HG-3 (DSMZ # ACC765), and CII (DSMZ #ACC773) are a chronic lymphocytic leukemia cell line. Su-DHL 6 (DSMZ #ACC572), U2932 (DSMZ # ACC633) and OCI-LY7 (DSMZ # ACC688) are a Diffuselarge B-cell lymphoma (DLBCL) cell line. JVM-2 (ATCC® CRL-3002) is amantle cell lymphoma cell line. BALL-1 (DSMZ # ACC742) is an acutelymphoblastic leukemia cell line.

Ramos cells in this in vitro model are indicative of how the combinationwill work in the treatment of non-Hogkins lymphoma (NHL) in humans. HG-3and CII cells in this in vitro model are indicative of how thecombination will work in the treatment of chronic lymphoid leukemia(CLL) in humans. Su-DHL 6, U2932 and OCI-LY7 cells in this in vitromodel are indicative of how the combination will work in the treatmentnon-Hodgkin's lymphoma in humans. JVM-2 cells in this in vitro model areindicative of how the combination will work in the treatmentnon-Hodgkin's lymphoma in humans. BALL-1 cells in this in vitro modelare indicative of how the combination will work in the treatment ofacute lymphoblastic leukemia in humans.

In summary, the combination of the exemplified anti-CD19 antibody andIbrutinib behaved synergistically in models relevant to CLL.

Therefore, the combination of the exemplified antibody specific for CD19and Ibrutinib should be effective in the treatment of humans innon-Hodgkin's lymphoma, chronic lymphocytic leukemia and/or acutelymphoblastic leukemia.

As the mechanism of action of Ibrutinib and other Bruton's tyrosinekinase (BTK) inhibitors are similar, as they all work by inhibiting thetyrosine-protein kinase BTK enzyme, which plays a crucial role in B-celldevelopment, it is believed that synergy should also be seen whentreating humans having non-Hodgkin's lymphoma, chronic lymphocyticleukemia and/or acute lymphoblastic leukemia with a combination of theexemplified anti-CD19 antibody and a Bruton's tyrosine kinase (BTK)inhibitor other than Ibrutinib.

As the exemplified anti-CD19 antibody and other anti-CD19 antibodiesbind CD19, it is believed that synergy should also be seen when treatinghumans having non-Hodgkin's lymphoma, chronic lymphocytic leukemiaand/or acute lymphoblastic leukemia with a combination of any anti-CD19antibody and a Bruton's tyrosine kinase (BTK) inhibitor, where theanti-CD19 antibody is, for example, described in U.S. patent applicationSer. No. 12/377,251 (Xencor), WO2005012493, WO2010053716 (Immunomedics);WO2007002223 (Medarex); WO2008022152 (Xencor); WO2008031056 (Medimmune);WO 2007/076950 (Merck Patent GmbH); WO 2009/052431 (Seattle Genetics);and WO2010095031 (Glenmark Pharmaceuticals), all of which areincorporated by reference in their entireties.

In embodiments, the antibody specific for CD19 comprises an antibodythat cross-competes with the antibody comprising an HCDR1 region ofsequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), anLCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region ofsequence MQHLEYPIT (SEQ ID NO: 6).

In embodiments, the antibody specific for CD19 comprises an antibodythat binds to the same epitope as an antibody comprising an HCDR1 regionof sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG(SEQ ID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3),an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region ofsequence MQHLEYPIT (SEQ ID NO: 6).

In embodiments, the antibody specific for CD19 comprises an HCDR1 regionof sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG(SEQ ID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3),an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region ofsequence MQHLEYPIT (SEQ ID NO: 6).

In embodiments, the antibody specific for CD19 comprises a variableheavy chain of the sequenceEVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWG QGTLVTVSS(SEQ ID NO: 10) and a variable light chain of the sequence

(SEQ ID NO: 11) DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYP ITFGAGTKLEIK.

In certain embodiments said antibody comprises a heavy chain constantdomain of the sequence

(SEQ ID NO: 12) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In embodiments, the antibody specific for CD19 comprises a light chainconstant domain of the sequence

(SEQ ID NO: 13) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC. 

In embodiments, the Bruton's tyrosine kinase (BTK) inhibitor isIbrutinib.

In embodiments, the components of the combination, the antibody specificfor CD19 and Ibrutinib, are administered separately. In an embodiment,Ibrutinib is administered prior to administration of the antibodyspecific for CD19.

In embodiments, the components of the combination are administered at atime where both components (drugs) are active in the patient at the sametime. It is implied by “synergism” that both drugs are active in thepatient at the same time. In embodiments, the components of thecombination are administered together, simultaneously, separately orsubsequently, either physically or in time. In embodiments, thecomponents of the combination are administered simultaneously.

In embodiments the combination is a pharmaceutical composition. Inembodiments, the composition comprises an acceptable carrier. Inembodiments, the combination is administered in an effective amount.

In another aspect the synergistic combination of an antibody specificfor CD19 comprising an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), anHCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region ofsequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequenceRSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS(SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6)and Ibrutinib is able to mediate killing of MEC-1 cells by ADCC in thepresence of isolated human PBMCs with an at least two-fold, three-fold,four-fold, or five-fold better efficacy than Ibrutinib alone.

An aspect of the present disclosure comprises a synergistic combinationof an antibody specific for CD19 comprising an HCDR1 region of sequenceSYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2),an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 regionof sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequenceRMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQID NO: 6) and Ibrutinib for the treatment of non-Hodgkin's lymphoma,chronic lymphocytic leukemia and/or acute lymphoblastic leukemia. Inembodiments, the non-Hodgkin's lymphoma is selected from the groupconsisting of follicular lymphoma, small lymphocytic lymphoma,mucosa-associated lymphoid tissue, marginal zone, diffuse large B cell,Burkitt's, and mantle cell.

In embodiments, the non-Hodgkin's lymphoma is follicular lymphoma. Inembodiments, the non-Hodgkin's lymphoma is small lymphocytic lymphoma.In embodiments, the non-Hodgkin's lymphoma is mucosa-associated lymphoidtissue. In embodiments, the non-Hodgkin's lymphoma is marginal zonelymphoma. In embodiments, the non-Hodgkin's lymphoma is diffuse large Bcell lymphoma. In embodiments, the non-Hodgkin's lymphoma is Burkitt'slymphoma. In embodiments, the non-Hodgkin's lymphoma is mantle celllymphoma.

In embodiments, the combination is for the treatment of chroniclymphocytic leukemia. In embodiments, the combination is for thetreatment of acute lymphoblastic leukemia.

Another aspect comprises a method of treating non-Hodgkin's lymphoma,chronic lymphocytic leukemia and/or acute lymphoblastic leukemia in anindividual in need thereof, which method comprises administration of anantibody specific for CD19 and a Bruton's tyrosine kinase (BTK)inhibitor. In embodiments of the method, the antibody specific for CD19comprises an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of sequenceGTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequenceRSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS(SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO:6). In embodiments of the method, the antibody comprises the exemplifiedantibody specific for CD19. In embodiments of the method the Bruton'styrosine kinase (BTK) inhibitor is Ibrutinib.

Another aspect includes a use of an antibody specific for CD19 whereinsaid antibody comprises an HCDR1 region of sequence SYVMH (SEQ ID NO:1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 regionof sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequenceRSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS(SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6)in the manufacture of a medicament for the treatment of non-Hodgkin'slymphoma, chronic lymphocytic leukemia and/or acute lymphoblasticleukemia in synergistic combination with Ibrutinib.

EXAMPLES Example 1: Cytotoxicity of MEC-1 Cells Using MOR00208 andIbrutinib Alone and in Combination Materials

Cell lines tested: MEC-1 cells (DSMZ # ACC497). Cell lines are tested:chronic B-cell leukemia cell line; JVM-2 (ATCC® CRL-3002) a mantle celllymphoma cell line; Ramos cells (ATCC number CRL-1596), a humanBurkitt's lymphoma cells; HG-3 (DSMZ # ACC765), and CII (DSMZ # ACC773)are a chronic lymphocytic leukemia cell line; Su-DHL 6 (DSMZ # ACC572),U2932 (DSMZ # ACC633) and OCI-LY7 (DSMZ # ACC688) are a Diffuse largeB-cell lymphoma (DLBCL) cell line; JVM-2 (ATCC® CRL-3002) is a mantlecell lymphoma cell line; and BALL-1 (DSMZ # ACC742) is an acutelymphoblastic leukemia cell line.

Culture conditions of cell lines used are according to supplier'sinformation.

Cell Medium: Iscove's Modified Dulbecco's Medium (IMDM), Invitrogen, CatNo.: 31980-048; RPM11640, Invitrogen, Cat No.: 31870-074; GlutaMAX,Invitrogen, CAT No.: 35050-38 LOT No.: 1504647; FCS: Sigma CAT No.:F7524 LOT No.: 111M3396.

NKs: RPM11640, with GlutaMAX™, Invitrogen, Cat No.: 31870-074, 10% FCS;Biocoll: Biochrome AG CAT No.: L6115 LOT No.: 0034D; MACS NK cellisolation kit: Miltenyi Biotec CAT No.: 130-092-657 LOT No.: 5150130115;Ibrutinib: Selleck Chem LOT No.: S2680; FCS: Sigma CAT No.: F7524 LOTNo.: 111M3396; and RefmAb33 (anti-RSV) with same Fc region as MOR00208.

Methods

The cytotoxicity of MOR00208 and Ibrutinib alone and in combination weretested in the MEC-1 cell line (CLL). The cytotoxicity of MOR00208 andIbrutinib alone and in combination are tested in the following targetcell lines: JVM-2, Ramos, HG-3, CII, Su-DHL 6, U2932, OCI-LY7, JVM-2 andBALL-1.

Ibrutinib is a covalent inhibitor of Bruton's tyrosine kinase and shouldabrogate proliferation in target cell lines. MOR00208 targets CD19 andmediates target cell killing via ADCC. Target cell killing is measuredusing the following parameters: Ibrutinib at a concentration range ofbetween 0.033 and 33 μM, specifically at 0.3 μM, 1.0 μM and 3.0 μM;MOR00208 at a concentration range from 0.001-10 nM, specifically at 0.01nM, 0.1 nM, and 10 nM, and the combination of MOR00208 and Ibrutinib.The following are used as controls: RefmAb33 or NK cells alone. In theIbrutinib group as well as in the MOR00208+Ibrutinib combination group,target cells are pre-treated with Ibrutinib for 7 days prior to the ADCCassay measurements. The target cells are counted and stained using 1μg/ml CFSE end concentration. For DMSO treated target cells, aneffector: target (E:T) ratio of 2:1 is chosen, corresponding to a celldensity of 5×10⁵/ml. The proliferative effect on target cells caused byIbrutinib treatment was included by adjusting the E:T ratio in inhibitortreated cells. The NK cells are counted and adjusted to 1×10⁶/ml. Thetarget cell killing assays were performed as follows: using 96 wellplates, 100 μl of target cell suspension was added per well, followed by100 μl cell suspension of NK cells to each well resulting in an E:Tratio of 2:1. The antibodies were diluted in a range of 10-0.001 nM inmedium. Cells were centrifuged and target: effector cell-pellets werere-suspended in 100 μl antibody-containing medium or the accordingcontrol solution. The assay was incubated for 4 h in CO2-incubator at37° C. After 10 min incubation on ice, 50 μl DAPI solution was added toeach well (final concentration 1 μg/ml) and incubated on ice for 10 min.The cell killing measurements were performed with FACS-Verse. Deadtarget cells were DAPI positive.

Pre-experiments following the methods described above were completedwith both MEC-1 cells and RAMOS cells. FIGS. 1-3 show the results of thepre-experiments.

Data

In total, three experiments were performed in order to determine themediation of ADCC on MEC-1 cells by the combination of MOR00208 andIbrutinib. The ADCC dose response curves for Experiments 1-3 are shownin FIGS. 6-8.

The percent (%) dead cells (raw data) for Experiments 1-3 are shown inTables 1-9 below.

Experiment 1

TABLE 1 Ibrutinib at 3 μM MOR00208 Concentration 10 nM 0.1 nM 0.01 nM A:MOR00208 alone 73.77 72.31 56.54 B: Ibr alone 3 μM 17.07 17.07 17.07 C:control 25.27 25.27 25.27 (0.1% DMSO/Ref33) AB: combination 88.01 86.5965.89

TABLE 2 Ibrutinib at 1 μM MOR00208 Concentration 10 nM 0.1 nM 0.01 nM A:MOR00208 alone 73.77 72.31 56.54 B: Ibr alone 1 μM 24.33 24.33 24.33 C:control 25.27 25.27 25.27 (0.1% DMSO/Ref33) AB: combination 85.48 84.8362.22

TABLE 3 Ibrutinib at 0.3 μM MOR00208 Concentration 10 nM 0.1 nM 0.01 nMA: MOR00208 alone 73.77 72.31 56.54 B: Ibr alone 0.3 μM 21.99 21.9921.99 C: control 25.27 25.27 25.27 (0.1% DMSO/Ref33) AB: combination80.51 77.08 56.79

Experiment 2

TABLE 4 Ibrutinib at 3 μM MOR00208 Concentration 10 nM 0.1 nM 0.01 nM A:MOR00208 alone 50.83 52.10 37.57 B: Ibr alone 3 μM 10.84 10.84 10.84 C:control 9.81 9.81 9.81 (0.03% DMSO/Ref33) AB: combination 66.70 62.4742.25

TABLE 5 Ibrutinib at 1 μM MOR00208 Concentration 10 nM 0.1 nM 0.01 nM A:MOR00208 alone 50.83 52.10 37.57 B: Ibr alone 1 μM 8.39 8.39 8.39 C:control (0.03% DMSO/Ref33) 9.81 9.81 9.81 AB: combination 63.56 63.9740.84

TABLE 6 Ibrutinib at 0.3 μM MOR00208 Concentration 10 nM 0.1 nM 0.01 nMA: MOR00208 alone 50.83 52.10 37.57 B: Ibr alone 0.3 μM 8.56 8.56 8.56C: control (0.03% DMSO/Ref33) 9.81 9.81 9.81 AB: combination 53.41 54.2234.83

Experiment 3

TABLE 7 Ibrutinib at 3 μM MOR00208 Concentration 10 nM 0.1 nM 0.01 nM A:MOR00208 alone 81.41 81.30 63.68 B: Ibr alone 3 μM 8.26 8.26 8.26 C:control (0.03% DMSO/Ref33) 16.40 16.40 16.40 AB: combination 86.19 86.3563.10

TABLE 8 Ibrutinib at 1 μM MOR00208 Concentration 10 nM 0.1 nM 0.01 nM A:MOR00208 alone 81.41 81.30 63.68 B: Ibr alone 1 μM 7.50 7.50 7.50 C:control (0.03% DMSO/Ref33) 16.40 16.40 16.40 AB: combination 88.75 85.5858.17

TABLE 9 Ibrutinib at 0.3 uM MOR00208 Concentration 10 nM 0.1 nM 0.01 nMA: MOR00208 alone 81.41 81.30 63.68 B: Ibr alone 0.3 μM 8.56 8.56 8.56C: control (0.03% DMSO/Ref33) 16.40 16.40 16.40 AB: combination 84.1782.49 55.89

Calculation of Synergism

The raw data (% dead cells) are analyzed as follows: 1) from the rawdata (% dead cells), the background (controls) are subtracted, resultingin the specific killing for each treatment group; then 2) the specifickilling values are normalized by setting the combination ofMOR00208+Ibrutinib to 1.

Combination Index (CI) calculations are completed in order to determinesynergy of the combination of the exemplified anti-CD19 antibody andIbrutinib as compared to MOR00208 and Ibrutinib alone. Such calculationsare described in Ting-Chao Chou, Theoretical Basis, Experimental Design,and Computerized Simulation of Synergism and Antagonism in DrugCombination Studies, Pharmacol Rev 58:621-681 (2006), which isincorporated by reference in its entirety and Chou T C, Talalay P,Quantitative analysis of dose-effect relationships: the combined effectsof multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22: 27-55(1984), which is incorporated by reference in its entirety. The methodsof Chou-Talalay are carried out using the CI-isobol method.

Median Effect Equation

The median-effect equation models the effect of an inhibitor (such as adrug) as F_(a)/F_(u)=(D/D50){circumflex over ( )}m, where D is the dose,F_(a) and F_(u) is the fraction of the system affected and unaffected bythe dose D (F_(a)+F_(u)=1); D50 is the dose producing the median effect(e.g. IC50, ED50, LD50). The constant m determines the shape of thedose-effect curve. We use GraphPad Prism to carry out a nonlinearregression calculation to estimate the parameters m and D50.

CI-Isobol Method

The CI-isobol method provides a quantitative assessment of synergismbetween drugs. A combination index (CI) is estimated from dose-effectdata of single and combined drug treatments. A value of CI less than 1indicates synergism; CI=1 indicates additive effect; and CI>1 indicatesantagonism. Drug interaction (synergism or antagonism) is morepronounced the farther a CI value is from 1.

Formally, the combination index (CI) of a combined drug treatment isdefined as

CI=D ₁ /D _(x1) +D ₂ /D _(x2)

Here D1 and D2 are the doses of drug 1 and drug 2 of the combination,respectively; and Dx1, and Dx2 is the dose of a treatment with only drug1 and drug 2 that would give the same effect as that of the combination.The doses Dx1 and Dx2 need to be estimated from the dose-effect data ofsingle drug treatments. Essentially, a median effect equation is fittedto the data of each drug. From the median effect equation of a drug, wecan estimate the dose (i.e. D) necessary to produce an effect (i.e.F_(a), F_(u)). The further a point lies from the additive line, thebigger the different between 1 and its CI, thus the stronger the(synergistic or antagonistic) effect is.

Results

The Chou index curves are shown in FIGS. 9-11. Data from the threeexperiments (at the same concentrations) was merged to produce one curvefor each Ibrutniib concentration.

The Chou index values indicate clear synergism of the combination ofMOR00208 and Ibrutinib in the specific killing of MEC-1 cells ascompared to MOR00208 and Ibrutinib alone.

Therefore, the combination of MOR00208 and Ibrutinib will also behavesynergistically in the treatment of non-Hodgkin's lymphoma (NHL),chronic lymphoid leukemia (CLL), and acute lymphoblastic leukemia (ALL)in humans.

Additional Analysis

Another approach to calculate and compare effects of single agents whenused in combination is the fractional product concept first described byWebb J. L. in “Enzymes and metabolic inhibitors” in 1963. This analysismethod considers that effects of several drugs can be directed againstthe same cell fraction, as long as the effects are mutuallynon-exclusive, which is true for MOR00208 and Ibrutinib, and thereforethe measured combination effect will be less as the theoreticalsummation of the single effects. The fractional product concept claims,whenever two drugs kill 50% of a target cell fraction the effect incombination would only be 75% (applied equation: 1−(1−0.5)×(1−0.5)=0.75)and not the expected 100%, due to only 50% of the target cells stillbeing viable and susceptible to one of the two drugs.

Another approach to calculate and compare effects of single agents whenused in combination is the approach of Clarke et al., Issues inexperimental design and endpoint analysis in the study of experimentalcytotoxic agents in vivo in breast cancer and other models, BreastCancer Research and Treatment 46:255-278 (1997), which is incorporatedby reference in its entirety.

The % dead cells (raw data) from Tables 1-16 was analysed in thefollowing way:

Antagonistic (AB)/C < (A/C) × (B/C) Additive (AB)/C = (A/C) × (B/C)Synergistic (AB)/C > (A/C) × (B/C)

where A is the treatment with MOR00208 alone; B is the treatment withIbrutinib alone; C is response to the control DMSO+RefMab33; AB is thecombination of treatments A and B.

Experiment 1

TABLE 10 Clarke analysis of Data shown in Table 1 MOR00208 Concentration10 nM 0.1 nM 0.01 nM (AB)/C 3.48 3.43 2.61 (A/C) × (B/C) 1.97 1.93 1.51

This Clarke analysis of the data shown in Table 1 is also graphicallyrepresented in FIG. 12.

TABLE 11 Clarke analysis of Data shown in Table 2 MOR00208 Concentration10 nM 0.1 nM 0.01 nM (AB)/C 3.38 3.36 2.46 (A/C) × (B/C) 2.81 2.76 2.15

This Clarke analysis of the data shown in Table 2 is also graphicallyrepresented in FIG. 13.

TABLE 12 Clarke analysis of Data shown in Table 3 MOR00208 Concentration10 nM 0.1 nM 0.01 nM (AB)/C 3.19 3.05 2.25 (A/C) × (B/C) 2.54 2.49 1.95

This Clarke analysis of the data shown in Table 3 is also graphicallyrepresented in FIG. 14.

Experiment 2

TABLE 13 Clarke analysis of Data shown in Table 4 MOR00208 Concentration10 nM 0.1 nM 0.01 nM (AB)/C 6.80 6.37 4.31 (A/C) × (B/C) 5.73 5.87 4.23

This Clarke analysis of the data shown in Table 4 is also graphicallyrepresented in FIG. 15.

TABLE 14 Clarke analysis of Data shown in Table 5 MOR00208 Concentration10 nM 0.1 nM 0.01 nM (AB)/C 6.48 6.52 4.16 (A/C) × (B/C) 4.43 4.54 3.27

This Clarke analysis of the data shown in Table 5 is also graphicallyrepresented in FIG. 16.

TABLE 15 Clarke analysis of Data shown in Table 6 MOR00208 Concentration10 nM 0.1 nM 0.01 nM (AB)/C 5.44 5.53 3.55 (A/C) × (B/C) 4.52 4.63 3.34

This Clarke analysis of the data shown in Table 6 is also graphicallyrepresented in FIG. 17.

Experiment 3

TABLE 16 Clarke analysis of Data shown in Table 7 MOR00208 Concentration10 nM 0.1 nM 0.01 nM (AB)/C 5.26 5.26 3.85 (A/C) × (B/C) 2.50 2.50 1.95

This Clarke analysis of the data shown in Table 7 is also graphicallyrepresented in FIG. 18.

TABLE 17 Clarke analysis of Data shown in Table 8 MOR00208 Concentration10 nM 0.1 nM 0.01 nM (AB)/C 5.41 5.22 3.55 (A/C) × (B/C) 2.27 2.27 1.78

This Clarke analysis of the data shown in Table 8 is also graphicallyrepresented in FIG. 19.

TABLE 18 Clarke analysis of Data shown in Table 9 MOR00208 Concentration10 nM 0.1 nM 0.01 nM (AB)/C 5.13 5.03 3.41 (A/C) × (B/C) 2.59 2.59 2.03This Clarke analysis of the data shown in Table 9 is also graphicallyrepresented in FIG. 20. Results of Clarke

Experiments 1-3 at each concentration showed clear synergy of thecombination of MOR00208+Ibrutinib using the methods of Clarke et al. Themethods of Clarke et al., however, show synergism despite in a fewexperiments where the Ibrutinib activity was less than that of thecontrol.

It is to be understood that the description, specific examples and data,while indicating exemplary embodiments, are given by way of illustrationand are not intended to limit the present invention. Various changes andmodifications within the present invention will become apparent to theskilled artisan from the discussion, disclosure and data containedherein, and thus are considered part of the invention.

We claim:
 1. A method for treatment of chronic lymphocytic leukemia,acute lymphoblastic leukemia or non-Hodgkin's lymphoma in a patient,said method comprising administering to the patient an antibody specificfor CD19 wherein said antibody comprises an HCDR1 region of sequenceSYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2),an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 regionof sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequenceRMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQID NO: 6) and a Bruton's tyrosine kinase (BTK) inhibitor.
 2. The methodaccording to claim 1, wherein the non-Hodgkin's lymphoma is selectedfrom the group consisting of follicular lymphoma, small lymphocyticlymphoma, mucosa-associated lymphoid tissue, marginal zone, diffuselarge B cell, Burkitt's, and mantle cell.
 3. The method according toclaim 1, wherein the non-Hodgkin's lymphoma is follicular lymphoma. 4.The method according to claim 1, wherein the non-Hodgkin's lymphoma issmall lymphocytic lymphoma.
 5. The method according to claim 1, whereinthe non-Hodgkin's lymphoma is mucosa-associated lymphoid tissue.
 6. Themethod according to claim 1, wherein the non-Hodgkin's lymphoma isdiffuse large B cell lymphoma.
 7. The method according to claim 1,wherein the non-Hodgkin's lymphoma is Burkitt's lymphoma.
 8. The methodaccording to claim 1, wherein the non-Hodgkin's lymphoma is mantle celllymphoma.
 9. The method according to claim 1, wherein the antibodyspecific for CD19 and the Bruton's tyrosine kinase (BTK) inhibitor ofsaid combination are administered separately.
 10. The method accordingto claim 1, wherein the Bruton's tyrosine kinase (BTK) inhibitor of saidcombination is administered prior to administration of the antibodyspecific for CD19.
 11. The method according to claim 1, wherein theantibody specific for CD19 and the Bruton's tyrosine kinase (BTK)inhibitor of said combination are administered simultaneously.
 12. Themethod according to claim 1, wherein the antibody specific for CD19 andthe Bruton's tyrosine kinase (BTK) inhibitor of said combination areadministered at a time where both drugs are active in the patient at thesame time.